Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 μg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and cause the life threatening disease botulism. Sensitive and broad detection is extremely challenging due to the toxins’ high potency and molecular heterogeneity with several serotypes and more than 40 subtypes. The toxicity of BoNT is mediated by enzymatic cleavage of different synaptic proteins involved in neurotransmitter release at serotype-specific cleavage sites. Hence, active BoNTs can be monitored and distinguished in vitro by detecting their substrate cleavage products. In this work, we developed a comprehensive panel of monoclonal neoepitope antibodies (Neo-mAbs) highly specific for the newly generated N- and/or C-termini of the substrate cleavage products of BoNT serotypes A to F. The Neo-mAbs were implemented in a set of three enzymatic assays for the simultaneous detection of two BoNT serotypes each by monitoring substrate cleavage on colour-coded magnetic Luminex-beads. For the first time, all relevant serotypes could be detected in parallel by a routine in vitro activity assay in spiked serum and food samples yielding excellent detection limits in the range of the mouse bioassay or better (0.3–80 pg/mL). Therefore, this work represents a major step towards the replacement of the mouse bioassay for botulism diagnostics.